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Abstract

While altered activities in sensory neurons were noticed in neuropathic pain, caused
by highly diverse insults to the peripheral nervous system, such as diabetes, alcohol
ingestion, cancer chemotherapy and drugs used to treat AIDS, other infections and
autoimmune diseases, as well as trauma, our understanding of how these various peripheral
neuropathies manifest as altered neuronal activity is still rudimentary. The recent
development of models of several of those neuropathies has, however, now made it possible
to address their impact on primary afferent nociceptor function. We compared changes
in mechanically-evoked C-fiber activity, in models of painful peripheral neuropathy
induced by drinking ethanol (alcohol) or administering 2',3'-dideoxycytidine (ddC),
a nucleoside reverse transcriptase inhibitor for AIDS therapy, two co-morbid conditions
in which pain is thought to be mediated by different second messenger signaling pathways.
In C-fiber afferents, ddC decreased conduction velocity. In contrast, alcohol but
not ddC caused enhanced response to mechanical stimulation (i.e., decrease in threshold
and increase in response to sustained threshold and supra-threshold stimulation) and
changes in pattern of evoked activity (interspike interval and action potential variability
analyses). These marked differences in primary afferent nociceptor function, in two
different forms of neuropathy that produce mechanical hyperalgesia of similar magnitude,
suggest that optimal treatment of neuropathic pain may differ depending on the nature
of the causative insult to the peripheral nervous system, and emphasize the value
of studying co-morbid conditions that produce painful peripheral neuropathy by different
mechanisms.

Background

The second messenger signaling pathways in primary afferent nociceptors that mediate
hypersensitivity to mechanical stimuli differ between models of painful peripheral
neuropathies [1]. Two extreme examples of this are the neuropathies induced by chronic ethanol consumption,
and by acquired immunodeficiency disease syndrome (AIDS) therapy (nucleoside reverse
transcriptase inhibitors). In alcohol-induced neuropathy, protein kinase Cε(PKCε)
has a major contribution to mechanical hyperalgesia [2], whereas in AIDS therapy neuropathy, Ca++, caspase signaling and mitochondrial electron transport [3-5] but not PKCε or a number of other second messenger signaling pathways (i.e., protein
kinase A, protein kinase G, extracellular signal-regulated kinases 1/2 or nitric oxide)
contribute [3].

Enhanced activity in sensory neurons is thought to contribute to pain reported by
patients with small-fiber peripheral neuropathies. Microneurography techniques have
demonstrated pathological responses such as sensitization to mechanical stimuli, in
patients with trigeminal neuralgia [6], traumatic nerve injury [7], entrapment neuropathy [8], phantom limb [9] and erythromelalgia [10]. However, there are practical limitations in performing microneurography in patients,
including inability to classify fiber functions fully, small numbers of fibers that
can be evaluated in an individual patient and the potential for inducing further injury
by introducing a microelectrode into an already damaged nerve. Furthermore, in spite
of the fact that in most patients, metabolic abnormalities, toxins, drugs or infectious
organisms are producing the neuropathic conditions, most microneurography studies
have been done in patients with a traumatic nerve injury [7-9].

Single-fiber electrophysiology has been performed in animal models of metabolic and
toxic, as well as traumatic nerve injury-associated painful peripheral neuropathy.
Following traumatic nerve injury it has been reported that there is increased spontaneous
activity occurring in irregular bursts [11-13]; in diabetic neuropathy, in addition to increased spontaneous activity, a decrease
in threshold and increase in response to supra-threshold stimulation has been reported
[14-19]; in models of cancer chemotherapy neuropathy, C-fibers have been reported to be hyperresponsive
and to fire irregularly [1,20]; in alcohol neuropathy, C-fibers also demonstrate a decrease in threshold and increased
response to stimulation [2]; and, in nucleoside reverse transcriptase inhibitor-induced AIDS-therapy neuropathy,
a change in post-stimulus interspike interval (ISI) histogram, without change in threshold
or number of action potentials in response to threshold or suprathreshold mechanical
stimulus has been reported [3]. In this study, we have performed a side-by-side comparison of evoked C-fiber activity
in models of two frequently co-morbid forms of peripheral neuropathy, alcohol and
AIDS therapy-induced painful peripheral neuropathy, which differ markedly in the nociceptor
second messenger signaling pathways involved [2,3].

Results

Conduction velocity

Conduction velocity, a measure of axonal excitability, has been used extensively in
the classification and diagnosis of peripheral neuropathies. The conduction velocity
of individual C-fibers, whose mechanical receptive fields had been identified, was
measured in sensory neurons innervating the dorsum of the hind paw of ethanol-consuming
and ddC-treated rats that demonstrated mechanical hyperalgesia prior to electrophysiology
study, and in control rats. While there was a decrease in conduction velocity in both
ethanol (decrease 11.7%) and ddC (decrease 16.4%) treated rats, the decrease was statistically
significant only in the AIDS therapy model (Figure 1, p < 0.05). Thus, as in patients with diverse forms of peripheral neuropathy who
have a decrease in conduction velocity in myelinated fibers, a decrease in the conduction velocity in C-fibers of rats with peripheral
neuropathy can also be shown. Since it is generally considered that slowed conduction
velocity is a manifestation of alterations in axonal ionic conductance [21], our findings are compatible with changes in ionic conductance in C-fiber axons in
AIDS therapy neuropathy. How such changes might contribute to symptoms associated
with ddC peripheral neuropathy requires further studies.

Figure 1. Mean conduction velocities (0.86 ± 0.03, 0.76 ± 0.04, 0.72 ± 0.03 m/sec) of C-fibers
from the three groups of rats (i.e., naive, ethanol (EtOH) and ddC, respectively)
were significantly different (one way ANOVA, p < 0.05). The conduction velocity of
C-fibers from the ddC group (n = 18) was significantly lower than that of control
rats (n = 38, p < 0.05) while the conduction velocity was similar between EtOH (n
= 15) and control groups (p > 0.05).

Response to mechanical stimulation

Threshold stimulus intensity

Alterations in primary afferent nociceptor function associated with enhanced pain
are thought to be due to a decrease in threshold for nociceptor activation and an
increase in number of action potentials fired. Therefore, the mechanical threshold
and response at threshold of C-fiber nociceptors was determined in control as well
as in alcohol-consuming and ddC-treated rats. In contrast to conduction velocity,
changes in most of the other electrophysiological parameters occurred only in the
rat model of alcohol-induced painful peripheral neuropathy. Thus, chronic ethanol
ingestion but not ddC administration produces a decrease in average C-fiber mechanical
threshold (43.5%; p < 0.05); in ddC-treated rats there was actually a small, not statistically
significant, increase in mechanical threshold (Figure 2).

Figure 2. The mechanical threshold of C-fibers in the EtOH group (n = 15) was significantly
lower that of control C-fibers (n = 38, p < 0.05, Mann Whitney test) while the mechanical
thresholds between ddC (n = 18) and control groups were similar (p > 0.05, Mann Whitney
test).

Response to sustained threshold and suprathreshold stimulus

Similar to their effects on C-fiber mechanical threshold, ethanol consumption but
not ddC administration significantly enhanced the number of action potentials fired
in response to sustained threshold and fixed suprathreshold (10 g) intensity mechanical
stimulation (Figure 3). These results provide further support for the suggestion that enhanced C-fiber
response contributes to the symptoms of alcohol-induced peripheral neuropathy and
raises the question of how the enhanced nociception in AIDS therapy painful peripheral
neuropathy is encoded. Thus, while our data support a role for enhanced C-fiber response
contributing to pain associated with alcohol neuropathy, it does not appear to contribute
in AIDS therapy neuropathy.

Figure 3. The mean responses to both sustained (60 sec) threshold and 10 g stimuli of ddC, EtOH
and control C-fibers were significantly different (one way ANOVA, p < 0.01). The responses
of EtOH group (n = 15) were significantly higher than those of control rats (n = 38,
p < 0.01, Tukey's multiple comparison test) while the responses of C-fibers in the
ddC group (n = 18) were similar to those of controls (p > 0.05, Tukey's multiple comparison
test).

Activity pattern

ISI

In spite of the ability of activity pattern to signal, independent of average firing
frequency [20,22], changes in activity pattern produced by various forms of painful peripheral neuropathy
has not been studied systematically. To analyze the changes in activity pattern generated
in response to stimulation of C-fiber nociceptors in the mechanical receptive field,
we first analyzed the ISI histograms for the response of C-fibers to sustained (60
sec) threshold and suprathreshold (10 g) mechanical stimulation, in ethanol-consuming,
ddC-treated and control rats. In alcohol consuming rats, the proportion of short ISIs
was significantly increased (p < 0.05, Figure 4B; p < 0.01, Figure 4E). A small, albeit statistically significant, increase in intermediate ISI was observed
with threshold stimulus, in ddC-treated rats (Figure 4C). That there is an increase in the number of short ISIs in rats with alcohol neuropathy,
suggests that temporal summation may play a role in the neuropathic symptoms associated
with alcohol consumption but not ddC treatment.

Figure 4. The ISI distributions of both EtOH (n = 15) group of C-fibers in responses to sustained
(60 sec) threshold and 10 g stimuli were significantly changed. A&D, the ISI distributions of control C-fibers (n = 38) in responses to sustained threshold
and 10 g stimuli, respectively. B&C, ISI 0.1–0.2 s of responses in EtOH group was significantly higher than that of controls
(p < 0.05, t-test) and ISI 0.3–0.4 s of responses in ddC group (n = 18) was significantly
higher than that of controls (p < 0.05, t-test). E&F, the ISI distributions of EtOH group in responses to 10 g stimulation were significantly
changed while the ISI distributions of ddC group were similar to those of controls.
ISI 0–0.2 s of responses in EtOH group was significantly higher than those of control
animals (p < 0.01, t-test)

Co-efficient of variability (Cv2)

Finally, changes in activity pattern, generated in response to mechanical stimuli,
was analyzed by determining the coefficient of variability (Cv2) distribution for
the response to sustained threshold and suprathreshold mechanical stimulation in C-fibers
from ethanol fed, ddC treated or control rats. The plots of Cv2 versus number of spikes,
in response to mechanical stimulation, for high-firing fibers in alcohol fed rats
were different from that of low-firing and control fibers (Figure 5). In these high-firing fibers, the maximum Cv2 values were less, such that there
were almost no occurrences of Cv2 values greater than 1.1, unlike the distribution
of Cv2 values in low-firing and control C-fibers. The variability of Cv2 values was
also smaller. These changes contrast with those observed in vincristine and diabetic
neuropathy [16,20], where high and variable Cv2 values were observed in high-firing fibers. In ddC-treated
rats there were no hi-firing C-fibers; the Cv2 distribution for C-fibers in ddC-treated
rats was similar to that for C-fibers from controls rats (Figure 5A&D).

Figure 5. The Cv2 distribution of high firing fibers (B, n = 7) for EtOH group is markedly different from low firing fibers (C, n = 8) for EtOH group and control animals (A, n = 38) while they were similar between low firing fibers for EtOH group and controls.
The Cv2 distributions were similar between ddC (D, n = 18) and controls.

Discussion

While it is generally accepted that enhanced activity in primary afferent nociceptors
plays an important role in the pain experienced by patients with peripheral neuropathy
[6-10], changes in activity in primary afferent nociceptors have received little attention,
including direct comparisons between changes in primary afferent nociceptor function
in different forms of painful peripheral neuropathy. In this study, we have compared
mechanically evoked C-fiber activity in rat models of alcohol and AIDS therapy-induced
peripheral neuropathy, for which enhanced nociception has been shown to be dependent
on different second messenger signaling pathways.

While the mechanical hyperalgesia observed in these two models of painful peripheral
neuropathy are of similar magnitude [2,3], the changes in C-fiber function differ markedly, being fairly well restricted to
a decrease in conduction velocity for AIDS therapy, while many aspects of mechanically-evoked
activity were effected by alcohol. Although clinical studies show slowed conduction
velocity in many types of peripheral neuropathy [21,23-25], the mechanism underlying slowing of conduction velocity remains to be established.
Available data suggest that changes in ionic currents, most especially for voltage-gated
ion channels, contribute to conduction velocity abnormalities [21,26,27]. The most well studied model with respect to mechanisms involved in changes in conduction
velocity is diabetic neuropathy [21], in which INa+, IK+ and Ih have been shown to decrease [26,27] and ICa2+ to increase [28-30]. However, depending on the composition of other ion channels in the membrane of the
sensory neuron, one may observe either enhanced or attenuated sensation [21]. Since slowing of nerve conduction velocity is the major change in C-fiber function
in the rat model of ddC-induced painful peripheral neuropathy, direct analysis of
ionic currents in dorsal root ganglion neurons treated with AIDS therapy could provide
important insights into the mechanisms involved in the pain associated with this class
of neuropathies.

While the relatively small change in single fiber electrophysiological properties
of primary afferent nociceptors observed in rats with ddC neuropathy might suggest
that changes in the peripheral terminal of sensory neurons make a minor contribution
to AIDS therapy-induced pain, we have previously shown that peripheral administration,
at the site of nociceptive testing, of antagonists of intracellular calcium [3], caspase signaling [4] and the mitochondrial electron transport chain [5], which in control animals have no effect on mechanical nociceptive threshold, reverses
ddC-induced mechanical hyperalgesia. Taken together these findings provide support
for the suggestion that changes in primary afferent nociceptor function, not tested
for in the present study, may play a role in the decreased behavioral mechanical nociceptive
threshold in the ddC-induced painful peripheral neuropathy. Alternatively, since the
mechanism of action of nucleoside reverse transcriptase inhibitor-induced neurotoxicity
is via their effects on mitochondrial function [31,32], it may be that a fraction of mitochondria are affected in most neurons, leading
to a smaller change in function in a larger percentage of sensory neurons. In contrast,
in alcohol, diabetic [16,17] and vincristine [20] peripheral neuropathy, the toxic insult appears to produce an all-or-none change
in activity, in a subset of neurons (i.e., the high-firing fibers) not observed in
AIDS therapy neuropathy.

Decrease in mechanical threshold and increase in number of action potentials elicited
by the same intensity stimulus contribute to inflammatory pain [33,34], which is characterized by mechanical hyperalgesia. In the present study we found
a decrease in mechanical threshold and increase in number of action potentials produced
by threshold and suprathreshold stimulation in rats consuming alcohol, but not in
ddC-treated rats. The increase in number of short ISIs, in response to both threshold
and suprathreshold mechanical stimulation, in alcohol fed rats, will increase temporal
summation in postsynaptic spinal dorsal horn neurons; increasing the range of ISIs,
near 100 ms, as observed in rats consuming alcohol, causes greater temporal summation
of C-fiber-evoked excitatory postsynaptic currents in dorsal horn neurons [35], and in the same range of ISIs, temporal summation of afferent activity appears to
be an important factor in human pain perception [36-41].

While pattern of activity in a presynaptic neuron can dramatically affect activity
in its postsynaptic neurons [42-46], much less attention has been given to the importance of the pattern of activity
elicited by mechanical stimulation of primary afferents in the pain associated with
peripheral neuropathy. In previous studies of painful peripheral neuropathy we have
observed that changes in primary afferent nociceptor function occur in an all-or-nothing
fashion. Thus, in models of diabetic [16,17] and vincristine [20] neuropathy, we found enhanced activity restricted to a subpopulation of C-fibers
(i.e., high-firing fibers), the function of the remaining C-fibers being similar to
those in control rats. In alcohol-induced neuropathy this dichotomy was also present.
Therefore, in our analysis of variability in action potential timing we also separately
evaluated the change in activity pattern in high- and low-firing C-fibers. Marked
alteration in the distribution of Cv2 values was observed in high-firing C-fibers
in alcohol-induced painful peripheral neuropathy; however, this change was different
from that in high-firing C-fibers in diabetic and vincristine-treated rats, in that
there was a marked decrease in maximum Cv2 in rats with alcohol-induced neuropathy.
While the mechanism underlying these changes is unknown, the lower Cv2 value can be
generated by a repetitively bursting pattern of activity [42]. The functional significance of variability in neuronal discharge patterns has been
the focus of study in somatosensory cortex and other sensory areas [42-44,46]. It has been suggested that such "variability may not be so much a flaw as a feature
that the brain puts to good use" to "provide the dynamic range for rapid modulation
of synaptic efficacy" [45]. This may be relevant to the function of nociceptors as afferent activity-dependent
plasticity in spinal nociceptive pathways is thought to be a crucial feature of pain
signaling [47], and may contribute to the progressive increase in pain during a prolonged stimulus,
even while adaptation decreases the mean firing frequency of nociceptive nerve fibers
[48].

In summary, in two models of painful peripheral neuropathies that differ markedly
based on the involvement of second messenger signaling mechanisms in primary afferents,
we have found marked differences in C-fiber activity. Our findings raise the question;
does activity in sensory neurons from different forms of peripheral neuropathy have
unique signatures? Since alcohol consumption and AIDS are common co-morbid conditions
[49-51], the possibility that they produce painful peripheral neuropathy by different mechanisms
raises the question are symptoms more severe in AIDS patients who chronically consume
alcohol? One step in developing an understanding of the importance of these mechanisms
would be to directly activate individual second messengers in primary afferent nociceptors,
to determine their effect on mechanically-evoked nociceptor activity, and then to
study specific ion channels in dorsal root ganglion neurons, in vitro, to determine the ionic basis of these differences. In vitro studies of the effect of ddC on specific ionic conductance may be especially important
in furthering our understanding of the functional alterations in AIDS therapy neuropathy,
which does not appear to markedly alter function of individual primary nociceptors.

Conclusion

Our results demonstrated that only ddC decreased conduction velocity of C-fiber afferents.
In contrast, alcohol but not ddC caused enhanced response to mechanical stimulation
and changes in pattern of evoked activity. Our data also support the suggestion that
different therapies are likely to be needed to effectively manage symptoms in different
forms of peripheral neuropathy.

Methods

Animal model

Male Sprague-Dawley Rats (280–420 g) from Bantin and Kingman (Fremont, CA, USA) were
used in these experiments. Animal care and use conformed to National Institutes of
Health (NIH) guidelines and was approved by the University of California at San Francisco
Committee on Animal Research.

Alcohol-induced painful neuropathy

The rats used in these experiments housed one per cage were fed Lieber-DeCarli liquid
diet (Dyets Inc., Bethlem, PA) containing ethanol (6.5% ethanol) [2,52-54] for 12 weeks. In this protocol, alcohol-induced hyperalgesia is well established
by the end of the seventh week and maximal between 8–12 weeks [2]. All rats demonstrated mechanical hyperalgesia prior to electrophysiology study.

2',3'-dideoxycytidine-induced neuropathy

The nucleoside reverse transcriptase inhibitor for AIDS therapies induces a painful
peripheral neuropathy in the rat [3]. A single dose of the AIDS therapy drug, 2',3'-dideoxycytidine (ddC, 50 mg/kg i.v.),
produces a significant reduction in nociceptive threshold from day 1 after its administration,
which persisted for more than 20 days [3]. Since the model produced mechanical hyperalgesia in 100% of animals [3], we did not perform behavioral studies for each of the animals used in the electrophysiology
experiments. Of note, this would only have the potential to underestimate the effect
of neuropathy on sensory neuron function.

Electrophysiology

In vivo single-fiber electrophysiology was performed, as previously described [16,18,20]. Briefly, rats were anesthetized with sodium pentobarbital (initially 50 mg/kg, i.p.,
with additional doses given throughout the experiment to maintain areflexia). At the
end of the experiment the rat was euthanized by pentobarbital overdose followed by
bilateral thoracotomy. Recordings were made from the saphenous nerve, which innervates
the dorsal surface of the hind paw. Bipolar stimulating electrodes were placed under
the nerve at a site distal to the recording site. The nerve was crushed proximal to
the recording site to prevent flexor reflexes during electrical stimulation of the
nerve. Fine fascicles of axons were dissected from the nerve and placed on a silver-wire
recording electrode. Single units were first detected by electrical stimulation of
the nerve. Receptive fields of identified C-fibers were located using a mechanical
search stimulus, either a blunt probe with smooth tip or a 60 g von Frey hair (VFH).
Each fiber's conduction velocity was calculated by dividing the distance between the
stimulating and recording electrodes by the latency of the electrically-evoked action
potential. Fibers that conducted slower than 2 m/s were classified as C-fibers [55,56]. The fiber was determined to be cutaneous if it was activated by lifting and stimulating
the skin and/or by moving skin with respect to its subcutaneous tissue. All C-fibers
employed in the present experiment had cutaneous receptive fields. The electrically
evoked action potential corresponding to the C-fiber whose receptive field had been
identified was verified by the latency delay technique, in which electrically evoked
spikes resulted in longer latency when the receptive field of the same fiber was stimulated
mechanically [57]. Mechanical threshold was determined with calibrated VFH and defined as the lowest
force that elicited ≥2 spikes within 1 s, in at least 50% of trials.

Sustained threshold mechanical stimulation was performed using a calibrated VFH that
was manually placed on the receptive field for 60 s. Sustained (60 s) suprathreshold
(10 g) mechanical stimulation was accomplished by use of a mechanical stimulator consisting
of a force-measuring transducer (Entran, Fairfield, NJ, USA) mounted in series with
interchangeable VFH filaments. Neural activity was stored using an IBM compatible
computer with micro 1401 interface (CED, Cambridge, UK) and further analyzed off-line
with Spike2 software (CED).

ISI analysis

ISI analysis was used to evaluate the temporal characteristics of the response of
C-fiber nociceptors to sustained mechanical stimulation, which was adopted from our
study of nociceptor activity in the rat model of vincristine-induced painful neuropathy
[20]. The ISIs for each C-fiber's response was grouped into 100 ms bins between 0 and
499 ms; ISIs greater than or equal to 500 ms were not analyzed [20]. This bin width also allows our data to be compared with that in other studies [35,58,59]. The number of intervals occurring in each bin was expressed as the percentage of
the total number of ISIs in the trial. This trial-by-trial normalization procedure
allowed the distribution of ISIs from several fibers to be averaged together.

Action potential firing variability (Cv2)

The coefficient of variability (Cv2) was calculated to compare the relative difference
between adjacent ISIs [42]. Cv2 is defined as the square root of 2 multiplied by the standard deviation of two
ISIs divided by their mean [42]. Thus, it is a dimensionless value that is independent of absolute firing rate. Based
on our previous studies in rat models of vincristine- and diabetes-induced peripheral
neuropathy [16,20], the response of each C-fiber during the 1 min duration of the stimulus was divided
into six consecutive 10 s periods, and the average Cv2 for all fibers in each corresponding
10 s period was calculated. Based on our similar finding in rat models of vincristine
and diabetes-induced painful peripheral neuropathy [16,20], fibers were divided into two groups, "low-firing" fibers which fired <100 spikes
and "high-firing" fibers which fired >100 spikes, so that the firing pattern of C-fiber
activity from the peripheral neuropathy models can be compared. The high-firing C-fibers
had approximately 2.5-fold higher responses to sustained threshold and suprathreshold
mechanical stimulation compared with control fibers during the 60 s stimulus while
the low-firing frequency C-fibers had responses similar to those of controls.

Statistics

Group data are expressed as mean ± S.E.M. Statistical analyses were done using analysis
of variance (ANOVA) followed by Tukey's multiple comparison post hoc test or unpaired
t-test and Mann Whitney U test, as appropriate. Differences were considered significant at P < 0.05.

List of abbreviations

ddC : 2',3'-dideoxycytidine

ISI : interspike interval

AIDS : Acquired Immunodeficiency Disease Syndrome

PKCε : protein kinase Cε

VFH : von Frey hair

Cv2 : coefficient of variability

EtOH : ethanol

Authors' contributions

XC participated in the design of the study, carried out all the experiment, performed
the statistical analysis and drafted the manuscript. JDL participated in the design
of the study and drafted the manuscript. All authors read and approved the final manuscript.

Acknowledgements

This research was supported by NIH grant NS21647. We thank Drs. Yinka Dina and Elizabeth
Joseph provided ethanol- and ddC- treated rats, respectively.

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